PROJECT SUMMARY In the adaptive immune response, cytotoxic T lymphocyte (CTL) continuously “crawl” seeking evidence of foreign peptide fragments on the surface of other cells. Once the T cell encounters a target cell with foreign or mutant peptides, then it is activated unleashing a potent immune response. Emerging evidence suggests that cell mechanical forces transmitted to the T cell receptor (TCR) contribute to its high specificity in antigen recognition and promote T-cell activation. This is not surprising, as the TCR and other T cell co-receptors bind their cognate ligands only when two dynamic cells physically “touch”. As a first step toward understanding the role of molecular forces in tuning T cell response, it is important that we measure the magnitude of forces transmitted to ligand- receptor complexes and then to relate mechanical events to signaling and functional responses. My PhD research (F99 phase) has focused on developing methods to measure and elucidate the role of mechanical forces in immune response. I have designed a microparticle tension senor that allows one to quantify receptor forces in high throughput and also to measure forces at curved cell junctions. Additionally, I used this assay to screen the dose-response function of drugs that modulate cell mechanics. Because T cell responses are fine tuned by an array of co-receptors, I tested the role of mechanics in LFA-1 function. In this work, I demonstrated that the magnitude of LFA-1 integrin forces fine tunes TCR triggered activation and antigen discrimination. In addition, I revealed mechanically active LFA-1 defines the permissive zones for cytotoxic secretion, and suppression of LFA-1 forces significantly abrogates cytotoxicity. My work suggests that receptors cooperate to tune T-cell responses. For the remainder of my F99 phase, I will investigate the mechano-communication between receptor forces. Specifically, I will develop a DNA origami nano device to pattern ligands and measure spatiotemporal colocalization of mechanical events. Afterwards, I will proceed to test this hypothesis on cell plasma membrane by engineering tension probes on the surface of living cells. This will enable one to control and measure TCR-forces at authentic cell-cell junctions that mimic the chemical and physical properties of the immune synapse. For my postdoctoral work (K00 phase), I aim to improve upon current cancer therapies by leveraging T cell mechanics in boosting the specificity of immune response. In adoptive cell therapy (ACT), after therapeutic T-cell reinfusion, adjuvant drugs such as cytokines need to be administered to boost immune reconstitution. However, nonspecific drug release causes side effects and T-cell exhaustion. To address this challenge, I will decorate T-cells with DNA cages that mechanically trigger the release of encapsuled drugs at the tumor zone. If successful, this work will significantly enhance the ACT efficiency and offer the first example that links m...